Detection of a variant form of high affinity receptor to IgE FCεRIβ using primers and probes

ABSTRACT

The invention relates to a method for diagnosing an individual as having atopy or a predisposition thereto. The method involves demonstrating in the individual the presence or absence of either: (i) an unusual variant form of the gene which codes for the beta sub-unit of the high affinity receptor for IgE (FcεRIβ), the variant form comprising a variation in exon (7) which encodes a glycine at amino acid residue 237 instead of glutamic acid which appears at residue 237 in individuals without atopy: or (ii) an unusual polymorphic form of the amino acid sequence for FcεRIβ, the polymorphic form comprising glycine at amino acid residue 237 instead of glutamic acid which appears at residue 237 in individuals without atopy. Materials and other methods relating to the above diagnostic method are also described.

The present invention relates to the diagnosis and therapy of atopy andto materials and methods relating thereto.

Atopy is in essence an excessive or misplaced immune response mediatedby IgE antibodies in response to common environmental allergens. Commonallergens sensitizing atopic individuals are grass and tree pollens,house dust, house dust mites (HDM), bacteria, fungi, feathers, hair,eggs, milk and chocolate. Atopy underlies hayfever, asthma, eczema,urticaria and certain gastro-intestinal disorders.

Asthma is an atopic condition of particular interest as it is becomingmore prevalent. It now affects one child in seven in the United Kingdom(Strachan D. P., et al., Arch. Dis. Childhood 70, 174-178, 1994).Allergies/atopy underlies 95% of childhood asthma cases. Asthma may beidentified by recurrent wheeze and intermittent airflow limitation. Anasthmatic tendency can be quantified by the measurement of bronchialhyper-responsiveness (BHR) in which an individual's dose-response curveto a broncho-constrictor such as histamine or methacholine isconstructed. The curve is commonly summarised by the dose which resultsin a 20% fall in airflow (PD20) or the slope of the curve between theinitial airflow measurement and the last dose given (slope).

In an atopic immune response, IgE is produced by B-cells in response toallergen stimulation. These antibodies coat mast cells by binding to thehigh affinity receptor for IgE (FcεRI). When a multivalent allergenbinds to an IgE coated mast cell, the cross-linking of adjacent IgEs byallergen initiates a series of cellular events leading to thedestabilization of the cell membrane and release of inflammatorymediators. This results in mucosal inflammation, wheezing, coughing,sneezing and nasal blockage.

Atopy can be diagnosed by (i) a positive skin prick test in response toa common allergen; (ii) detecting the presence of specific serum IgE forallergen; or (iii) by detecting elevation of total serum IgE. Usingthese and other assays, a genetic linkage between generalised atopic IgEresponses and chromosome 11q has been observed.

Previous studies have found linkage of atopy and bronchialhyper-responsiveness to markers on chromosome 11q13 (eg Cookson, W. O.C. M., et al., 1989 Lancet i pages 1292-1295.) and the beta-chain of thehigh affinity receptor for IgE (FcεRIβ) has been identified as acandidate gene for this linkage (Sandford, A. J., et al., 1993 Lancet341, pages 332-334).

WO95/05481 provides further information on atopy and its diagnosis andtreatment. In particular, it discloses variants of the gene encodingFcεRIβ which are associated with atopy and it teaches a method fordiagnosing atopy which is based upon the demonstration of the presenceor absence of one or two variants in a specific portion of the DNAsequence of the gene encoding FcεRIβ. The specific DNA sequence islocated near the commencement of exon 6 of the FcεRIβ gene on chromosome11q and two variants are described. The first variant (designated I181L)encodes a polymorphism in which there is a change of isoleucine toleucine at amino acid residue 181. The second variant (designatedI181L/V183L) involves a double mutation that includes the change atresidue 181 and the additional change of valine to leucine at residue183. The amino acid residues 181 and 183 are positioned in the fourthtransmembrane domain of FcεRIβ. The variants and polymorphisms are fullydescribed in WO95/05481.

The polymorphisms I181L and I181L/V183L and associated polynucleotidevariants have been described in British and Australian populations andare, in selected subjects, associated with atopy and atopic asthma(Shirakawa, T., et al. 1994 Nature Genetics 7, 125-9; and Hill, M. R. etal., 1995 Br. Med. J., 311, 776-779). However the twopolymorphisms/variants I181L and I181L/V183L have in practice provedproblematical to assay, there being a high false-negative rate forPCR-based tests.

Unexpectedly, the present applicants have found a further variantassociated with atopy and atopic asthma which is in the coding sequencefor the C-terminal cytoplasmic tail of FcεRIβ (the first quarter of theC-terminal tail is encoded in exon 6 and the remainder in exon 7). Thisnew variant (designated E237G) is located within exon 7 of the FcεRIβgene. The normal sequence of the FcεRIβ gene has been published inKuster, H., et al. 1992, J. Biol. Chem., 267, 12782-12787, "The gene andcDNA for the human high affinity immunoglobulin E receptor β chain andexpression of the complete human receptor". The sequence can also befound in the Genbank/Embl Databases under Accession No. M89796.

The newly discovered variation in the coding sequence for the C-terminalcytoplasmic tail of FcεRIβ results in a change of glutamic acid toglycine at amino acid residue 237. Sequencing of exon 7 in atopicindividuals confirms a nucleotide change from adenine to guanine atnucleotide residue 6843 of the FcεRIβ gene (Kuster et al., 1992supra.,). For completeness it should be noted that nucleotide residue6843 of the 1992 Kuster et al sequence, corresponds to nucleotideresidue number 7297 in the Genbank/Embl Database sequence.

To data the applicants have investigated 1004 Australian Caucasianindividuals in 230 nuclear families for the presence of the E237Gpolymorphism. 26 males and 26 females were found to have thepolymorphism giving a population prevalence of about 5.3%. The E237Gpolymorphism was significantly associated with increases in (i) skintest responses to house dust mite (p=0.02 by Mann-Whitney U test) andgrass pollen (p=0.0008); (ii) radioallergosorbent test specific IgEtitres (RAST) to HDM (p=0.001) and grass pollen (p=0.04); and (iii) BHRto metacholine (p=0.0006). The relative risk of children carrying theE237G polymorphism having asthma was 2.4 compared to children withoutthe variant (95% CI 1.29-4.32; p=0.004). Unlike the I181L andI181L/V183L polymorphisms which are maternally linked, the E237Gpolymorphism is independent of the parental origin of the allele.

In a similar study of individuals in Japan, the E237G polymorphism hasalso been found to be associated with atopic asthma, there being aparticularly high association with childhood asthma (Shirakawa, T. etal., 1996 Human Molecular Genetics, Vol. 5, No. 8 pages 1129 to 1130.).

The high affinity receptor for IgE, FcεRI, is a tetrameric complexformed by an α chain which can bind IgE, a β chain and a homodimer of γchains. It belongs to a family of receptors that contain homologousactivation motifs that appear capable of autonomously triggering cellactivation via protein-tyrosine phosphorylation. Two forms of theactivation motif appear in FcεRI: one in the β chain and one in the γchain. They are believed to operate synergistically. Upon triggering ofFcεRI, the protein-tyrosine kinase Lyn, already associated with the βchain becomes activated and phosphorylates the β and γ chains.Phosphorylation of the γ chain induces the association ofprotein-tyrosine kinase Syk with the γ chain and the subsequentactivation of Syk that then activates phospholipase (C.sup.γ 1 leadingto calcium mobilization). The β chain cannot initiate the fullsignalling capacity of FcεRI. However the importance and unique role ofthe β chain is demonstrated when the cytoplasmic C-terminal tail isremoved with the consequence that signalling is abolished even when theγ dimer is present. This indicates a role for the β chain in controllingreceptor phosphorylation. Amino acid residue 237 (the site of thepolymorphism) is located adjacent to the immunoreceptor tyrosineactivation motif (residues 212-228) reported in the cytoplasmicC-terminal tail of FcεRIβ. The polymorphism E237G introduces asignificant hydrophobicity change within the C-terminus of FcεRIβ whichmay effect the intracellular signalling capacity of FcεRIβ. Thesubstitution of the polar negatively charged glutamic acid at residue237 with the smaller polar, but uncharged, glycine results in the lossof the hydrophilic nature of this region when examined by theChou-Fassman hydrophobicity prediction program (GCG Molecular BiologyPackage, Wisconsin).

Thus the present applicants have unexpectedly established that there isa previously unrecognised variant/polymorphism (in the nucleotide/aminoacid sequences respectively) in the cytoplasmic C-terminal tail ofFcεRIβ which comprises sequence changes associated with a change ofglutamic acid to glycine at amino acid residue 237 and which isassociated with atopy and atopic asthma. These new and unexpectedteachings make possible new diagnostic methods.

Thus the present invention provides a method for diagnosing anindividual as being atopic or asthmatic, or of having a predispositionto atopy or asthma which comprises demonstrating in the individual thepresence or absence of an unusual variant form of the gene which codesfor FcεRIβ, the variant form encoding a polymorphism comprising glycineat residue 237 instead of glutamic acid.

As an alternative to analysing the gene for a variant, the amino acidsequence may be analysed for the equivalent polymorphism.

Thus the present invention also provides a method for diagnosing anindividual as being atopic or asthmatic or of having a predisposition toatopy or asthma which comprises demonstrating in the individual thepresence or absence of an unusual polymorphic form of the amino acidsequence for FcεRIβ, the polymorphic form comprising a glycine atresidue 237 instead of glutamic acid.

The presence or absence of an unusual polymorphic form of the amino acidsequence for FcεRIβ may be determined by use of a substance whichcomprises an antibody binding domain which specifically/selectivelybinds to the unusual polymorphic form which comprises glycine at residue237 instead of glutamic acid, but not to the normal form of FcεRIβ whichoccurs in the individuals without symptoms of atopy/asthma. Thesubstance may comprise an antibody, which may be polyclonal ormonoclonal.

Where analysis is made at the genetic level, this may be done byemploying a technique using one or more nucleic acid sequences which areable to hybridise to the unusual variant form of the gene under suitablestringency conditions. Typically, one or more of such sequences may beused as primers in a detection system comprising a polynucleotideamplification technique such as PCR, ARMS.

The amplification primers may be specific to the unusual variant, butnot to wild-type. In which case amplification will only occur if theunusual variant is present in the test sample. Alternatively, theamplification primers may be non-specific. In which case they are usedto amplify a sequence region of the variant gene which comprises thenucleotide change(s) which effect the amino acid change at residue 237.Thus an unusual variant such as E237G may be detected by specific PCRusing the established techniques of direct sequencing, SSCP and ARMS(Sanger F., et al., 1977 PNAS USA 74: 5463-5467; Orita M., et al., 1989Genomics 5: 874-879; Newton C. R., et al., 1989 Nucl. Acids Res., 17:2503-2516).

PCR, ARMS etc may be followed by direct sequencing and sequenceanalysis, analysis of single stranded conformational polymorphism (SSCP)and/or size analysis of the amplification products where theamplification reaction is designed to amplify a nucleotide sequence thesize of which is characteristic of the presence of the unusual variantcomprising the nucleotide changes and/or by probing the amplificationproducts with a sequence-specific nucleic acid probe capable ofannealing to a portion of the amplified gene which is specific to theunusual variant and not to wild-type. Any suitable method for theconfirmation of the existence of a particular nucleotide sequence may beemployed and the suggestions given above are for the purposes ofillustration only.

DNA or RNA-based amplification methods may be employed. Thus theamplification may be carried out based on mRNA or cDNA made from mRNA.

If primers used are specific to the variant, there will only besuccessful amplification by eg PCR, ARMS if the variant is present inthe test sample. Alternatively the primers used could be non-specific tothe variant, the amplification serves only to increase sample materialfor analysis by a different method such as direct sequencing and SSCP asmentioned above. Suitable nucleic acid sequences for use as specificprobes, or specific or non-specific primers in a detection assay forE237G variant/polymorphism as discussed herein can be ascertained fromknowledge of the known DNA sequence (Kuster et al., 1992 supra.) and thedisclosure of the existence of the variant/polymorphism as providedherein and come within the scope of the present invention.

The teaching of the existence of the E237G variant/polymorphism enablesone skilled in the art to easily design and make in accordance withknown techniques eg probes and primer pairs based on the publiclyavailable sequence information for FcεRIβ. Similarly a skilled personwould be able to ascertain from the publicly available sequenceinformation for FcεRIβ, peptides and polypeptides which may be made andemployed in accordance with known techniques, to produce substancescomprising an antibody binding domain which specifically/selectivelybind to the E237G polymorphic form and fail to bind substantially to thenormal form of FcεRIβ which occurs in individuals without symptoms ofatopy/asthma.

The patient sampling and assay system (whether assaying polynucleotideor amino acid sequences) may be in accordance with standard techniquesin the art. However such assay systems will be newly utilisingcomplementary nucleic acid sequences as probes or primers, or specificantibodies identified by the applicants by virtue of the disclosure ofthe E237G variant/polymorphism made herein as being useful for thediagnosis of atopy or asthma or a predisposition there towards.

Thus the present invention also provides oligonucleotide hybridisationprobes which comprise a sequence of nucleotides with sufficientcomplementarity and length to enable specific hybridization undersuitably high stringency conditions, to a portion of test sample geneticmaterial deriving from an individual patient which comprises thevariation in the sequence which gives rise to an unusual E237G form ofthe gene for the beta sub-unit as discussed above; the probe itself mayhybridise to the unusual sequence of nucleotides which can give rise tothe variant gene. The oligonucleotide hybridisation probe may not showsignificant hybridisation to an equivalent portion of test samplegenetic material derived from an individual without symptoms ofatopy/asthma.

A probe should comprise sufficient nucleotides for efficient andspecific annealing in order that the diagnostic analysis be sufficientlyaccurate. Typically a probe may comprise 17 to 20 nucleotides, but ofcourse the skilled person could prepare probes of other lengths and testthem in accordance with known techniques and the disclosures herein forfunctionality in a given set of suitably high stringency hybridisationconditions. In order to aid detection of specific hybridisation, theprobe may be labelled in accordance with techniques well-known in theart.

In the alternative, there are provided nucleic acid sequences withsufficient complementarity and length to enable specific hybridizationunder suitably high stringency conditions to a portion of test samplegenetic material which comprises the variation in the sequence whichgives rise to the E237G form of the gene for the beta sub-unit asdiscussed above, for use as primers in a reaction to amplify the gene.Typically there may be provided a pair of such primers. Theamplification reaction may comprise one stage of a diagnostic assay. Thehybridization of such primers under suitably high stringency conditionsmay be to a portion of nucleotide sequence lying in the vicinity of, butupstream and/or downstream of the E237G variant nucleotides which giverise to the unusual form of FcεRIβ. The primers may be non-specific forthe E237 variant nucleotides or a primer may be specific for the E237variant nucleotides. The methods as herebefore described may utilisenucleic acid sequences, probes and primers both as generally describedabove, or of the specific examples (or functional equivalents thereof)given in the detailed description which follows.

Reference above is made to high-stringency conditions for hybridization.Hybridization is the process whereby two single-stranded polynucleotidesanneal to each other to form a double-stranded molecule. The annealmentis effected by hydrogen bonding between the complementary bases in thetwo strands. However under certain reaction conditions (non-stringentconditions) any pair of single-stranded polynucleotides will anneal toeach other to form a double-stranded molecule (non-specifichybridization). Under different reaction conditions (high stringencyconditions) this non-specific hybridization is reduced or minimised sothat hybridization occurs, only between a pair of single-strandedpolynucleotides with substantial (if not complete) complementarity toeach other. The more stringent the conditions are, the greatercomplementarity is needed for hybridization. As stringency conditionsare decreased, so does the requirement for complementarity.

Thus the skilled person will in accordance with standard techniquesidentify a set of reaction conditions sufficiently stringent for theirparticular purpose. Generally of course the aim is to identify by use ofone single-stranded polynucleotide sequence as a probe or primer anothersingle-stranded complementary sequence in a test sample which is eitherexactly complementary or substantially complementary to the probe orprimer sequence. The reaction conditions (the high stringency condition)at which the hybridization is adequately specific will also bedetermined by the length of the single-stranded polynucleotide sequenceused as a primer or probe. Where the primer/probe sequence is veryshort, greater care may be needed in order to avoid non-specific bindingand the reaction conditions therefore may need to be highly stringent.The reaction conditions however may need to be less stringent where theprimer/probe sequence is long (and therefore likely to be veryspecific).

It can be seen from the above that `high-stringency conditions` arevariable according to the nature of the primer/probe and whether or notthe skilled person is desirous of identifying only exactly complementarysequences or of sequences with incomplete complementary. The skilledperson in accordance with standard techniques is able to identify forthemselves the high stringency conditions appropriate to theirexperimental or diagnostic aim.

Analysis may also be made of the amino acid sequence. This may be doneby sequencing studies, or by use of a probe comprising an antibodybinding domain which is specific for the E237G polymorphism. The probesmay comprise antibodies (monoclonal or polyclonal) which may be raisedagainst the unusual E237G polymorphism eg by use ofpeptides/polypeptides ascertained and made in knowledge of the availablesequence information for FcεRIβ as mentioned above. The probes may belabelled in accordance with standard methodologies. In vitro or in vivodiagnosis may be possible. Thus the present invention also providesprobes comprising an antibody binding domain which are specific for theE237G polymorphism. Such `antibody binding domain` probes may not bindto the normal form of FcεRIβ present in individuals without symptoms ofatopy/asthma. The probes may be antibodies. They may be polyclonal ormonoclonal.

As mentioned above, given knowledge of the amino acid sequence forFcεRIβ and the unusual E237G polymorphic forms associated with atopy orasthma described herein, standard synthesis techniques may be used tomanufacture short peptides or longer peptides characteristic of theE237G polymorphism and not of the normal `wild-type` form in individualswithout atopy, for use as immunogens to raise antibodies. In thealternative, the E237G polymorphism associated with atopy or asthma anddescribed herein may be isolated and purified for use as an immunogen toraise antisera. The present invention also therefore providespolypeptides (synthetic or isolated) and immunologically cross-reactivederivatives and fragments eg short peptide sequences which arecharacteristic of the E237G polymorphism as specifically describedtherein.

The present invention also provides diagnostic kits and reagents for thedetection of atopy or asthma or a predisposition there towards,comprising any of nucleic acid sequences, probes, primers, antibodies,polypeptides or peptides as described above. Also provided is the use ofany of nucleic acid sequences, probes, primers, antibodies, polypeptidesor peptides as described above in methods as described, or in themanufacture of diagnostic kits and reagents for the detection of atopyor asthma or a predisposition there towards.

In terms of treatment possibilities the disclosure that the E237Gpolymorphic form of the cytoplasmic C-terminal tail of FcεRIβ isassociated with atopy or asthma. enables assays to screen for compoundswhich down-regulate the unusual polymorphic form. The sequenceinformation provided herein will also enable the development ofantisense RNA treatment strategies to block the E237G polymorphism ofthe cytoplasmic C-terminal tail of FcεRIβ. Antibody based therapiesfocussing on the E237G polymorphism may also be possible. Use may bemade of an antibody, possibly humanised in accordance with knowntechniques, or of a compound comprising just an antibody domain which isspecific for the E237G polymorphism for treatment of an individual withatopy or asthma or a predisposition there towards. In particular, theeffect of the unusual polymorphic form of the cytoplasmic C-terminaltail of FcεRIβ on intracellular signalling may identify a novel targetfor therapeutic intervention.

Thus the present invention also extends to cover use of any of nucleicacid sequences, probes, primers, antibodies, polypeptides or peptides asdescribed above in treatment strategies or in methods to identifycompounds of prophylactic or therapeutic value. Invention also extendsto any such prophylactic or therapeutic compounds identified by use ofthe information materials and methods disclosed herein.

In summary, detection of an unusual E237G polymorphic form of the betasub-unit of FcεRIβ or detection of a coding sequence for said E237Gpolymorphism is a new and unexpected finding of diagnostic use inpredicting/diagnosing atopy and specific atopic conditions such asasthma. If an E237G polymorphism/variant associated with atopy or asthmais detected in an individual preferably at the neonatal stage inaccordance with the teachings of the present application, strategies forprevention of atopy or asthma by environmental manipulation to controlcontact with common allergens and/or vaccination against commonallergens can then be devised for the individual concerned.

Furthermore, the E237G polymorphism may also define a subgroup of egasthma sufferers with a particular clinical course, in which caserecognition of the variant/polymorphism would be of value in definingprognosis and management of asthma.

There now follows a more detailed description about testing for theE237G variant in FcεRIβ.

Located in exon 7, E237G lies within the C-terminal cytoplasmic tail ofFcεRIβ. The polymorphism is an amino acid change at residue 237 fromglutaminc acid to glycine. The polymorphism E237G is detected by egspecific PCR using the established techniques of direct sequencing, SSCPand ARMS.

(A) Direct sequencing and Single Stranded Conformational Polymorphism(SSCP)

SSCP is a technique in which DNA is amplified and then split into itstwo component chains and then moved down a gel by electrophoresis. Thepresence of a mutation on variant/polymorphism in one of the chains maymean that it forms a different shape (conformation) in the gel and somoves separately from the wild type thereby allowing detection.

A sample of patient blood is taken and peripheral blood leucocytesobtained in accordance with standard techniques. DNA is extracted fromthe leucocytes by standard techniques (see eg Blin, N., et al., 1976Nucleic Acids Research., Vol. 3, page 2303: "A general method forisolation of high molecular weight DNA from eukaryotes").

Direct sequencing and SSCP involves a two round PCR (two rounds is notessential, but it provides a cleaner product) with a common first round.For direct sequencing the second round incorporates a biotinylatedprimer.

Schematically: ##STR1##

    __________________________________________________________________________    Primer pairs                              Product size                        __________________________________________________________________________    1st round        5'                       3'                                                       B7F1:   CCA GCT AGT CTG GTT TGG TTT   (SEQ ID NO:1)                                                515 bp                                                   B7F2:   ATT AAG GTG GAC AGA AGC AGC   (SEQ ID NO:2)                                                  - 2nd round                         5'-3' sequencing  B7S1:   GAT GAG GTA AGT CTC TTG AG    (SEQ ID NO:3)                                                 238 bp                                                 *B7B2:   AAC CTT GGC CTT CTG GAT       (SEQ ID NO:4)                                                  3'-5' sequencing *B7B1:   TGA                                                GGT AAG TCT CTT GAG       (SEQ                                                ID NO:5) 238 bp                                         B7S2:   CAA AAC CTT GGC CTT CTG G     (SEQ ID NO:6)                                                   - SSCP              B7S1:                                                   GAT GAG GTA AGT CTC TTG AG                                                    (SEQ ID NO:7) 240 bp                                    B7S2:   CAA AAC CTT GGC CTT CTG G     (SEQ ID             __________________________________________________________________________                                              NO:8)                                (*5' biotinylated primer)   PCR reaction mixture                         

1st round 10 μl per reaction volume: Template is 50-100 ng genomic DNA.

2nd round 50 μl per reaction volume: Template is 1 μl 1st round productdiluted ×100 in dH₂ O.

Reaction mixture for 1st and 2nd round: 0.1 μg each primer, 200 μM dNTP,50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl₂ and 0.2 units Taqpolymerase (Boehringer Mannheim).

PCR conditions

    ______________________________________                                        1st round          2nd round                                                  ______________________________________                                        94° C. 5.0 minute 1 cycle                                                                 94° C. 5.0 minute 1 cycle                             94° C. 1.0 minute 94° C. 1.0 minute                             59° C. 1.0 minute 35 cycle 50° C. 0.5 minute 20 cycle                             72° C. 1.0 minute 72° C. 0.5 minute                             72° C. 10.0 minute 1 cycle 72° C. 10.0                         minute 1 cycle                                             ______________________________________                                    

(Ai) Direct sequencing

Preparation of single strand template DNA

Single strand template is prepared from 40 μl 2nd round PCR productusing streptavidin coated magnetic beads (Dynabeads, Dynal UK Ltd)following manufacturer's instructions.

Sequencing

Performed using Sequenase Version 2.0 DNA Sequencing Kit (Amersham LIFESCIENCE) and [α-³⁵ S] dATP radionucleotide following suggested protocol.Sequencing primer used is the non-biotinylated primer of the 2nd roundPCR. Sequencing in either direction detects E237G and it is sometimesuseful to perform both as confirmation when heterozygote sequenceappears faint.

(Aii) SSCP

Using Bio-Rad Protean II Cell apparatus (Bio-Rad Laboratories Ltd, UK),4 μl of second round PCR product was electrophoresed at 10 W per gel at4° C. for 22 hours in a 10% (w/v) polyacrylamide (19: 1 acrylamide:bis)/10% (v/v) glycerol gel with 1× TBE buffer. DNA was visualized bysilver staining using a Bio-Rad Silver Stain Kit (Bio-Rad LaboratoriesLtd, UK) and then gels were vacuum-dried onto Whatman 3M paper (MerckLtd).

(B) ARMS

Primers

B7FA1 (SEQ ID NO: 9): TGG CCA GCT AGT CTG GTT TGG TTT TCT GGA

B7FA2 (SEQ ID NO: 10): GGA GCA TAT TAA GGT GGA CAG AAG CAG CAG

B7M1 (SEQ ID NO: 11): ATT CAG CTA CTT ACA GTG AGT TGG AAG ACC CAG GCG G

B7W2 (SEQ ID NO: 12): CAC GTG ATT CTT ATA AAT CAA TGG GAG GAG ACA ATT

Schematically: ##STR2## PCR reaction mixture 50 μl reaction volume:0.1-0.2 μg genomic DNA, 250 ng of primers B7FA1, B7FA2, B7W2 and 125 ngof primer B7M1, 200 μM dNTP, 50 mM Kcl, 10 mM Tris-HCl (pH 8.3), 1,5 mMMgCl₂ and 0.2 units Taq polymerase (Boehringer Mannheim).

PCR conditions

A Hot start is recommended, Taq polymerase being added in 20% of thefinal volume of PCR reaction mixture after the first cycle which is heldat 80° C.

94° C. 5.0 minute 1 cycle (Hold at 80° C., add Taq)

94° C. 1.0 minute

60° C. 2.0 minute 35 cycle

72° C. 2.0 minute

72° C. 10.0 minute 1 cycle

Genotyping

Amplified product is visualized in an ethidium stained 4% agarose gel(3.0 g NuSieve* GTG (Flowgen), 1.0 g LMP agarose (GIBCO BRL), 50 μgethidium bromide per 100 ml 0.5× TBE) run for two hours at 60 V. Inaddition to the 446 bp control band, if present wild type sequence willresult in a 280 bp band and E237G sequence in a 238 bp band.

For any additional general guidance on interpreting the detailedinstruction given above, reference may be made to Sanger F. et al., 1977supra., Orita M., et al., 1989 supra and Newton C. R., et al., 1989supra., or to standard texts such as Short Protocols in MolecularBiology Second Edition A Compendium of Methods from Current Protocols inMolecular Biology edited by Frederick M. Ausubel et al. and MolecularCloning, A Laboratory Manual eds Sambrook, Fritsch and Maniatis, ColdSpring Harbor Laboratory Press, 1989.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 12                                       - - <210> SEQ ID NO 1                                                        <211> LENGTH: 21                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: Primer        - - <400> SEQUENCE: 1                                                         - - ccagctagtc tggtttggtt t           - #                  - #                      - #21                                                                   - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 21                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: Primer         - - <400> SEQUENCE: 2                                                         - - attaaggtgg acagaagcag c           - #                  - #                      - #21                                                                   - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: Primer         - - <400> SEQUENCE: 3                                                         - - gatgaggtaa gtctcttgag            - #                  - #                      - # 20                                                                   - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: Primer         - - <400> SEQUENCE: 4                                                         - - aaccttggcc ttctggat             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 5                                                   <211> LENGTH: 18                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: Primer         - - <400> SEQUENCE: 5                                                         - - tgaggtaagt ctcttgag             - #                  - #                      - #  18                                                                   - -  - - <210> SEQ ID NO 6                                                   <211> LENGTH: 19                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: Primer         - - <400> SEQUENCE: 6                                                         - - caaaaccttg gccttctgg             - #                  - #                      - # 19                                                                   - -  - - <210> SEQ ID NO 7                                                   <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: Primer         - - <400> SEQUENCE: 7                                                         - - gatgaggtaa gtctcttgag            - #                  - #                      - # 20                                                                   - -  - - <210> SEQ ID NO 8                                                   <211> LENGTH: 19                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: Primer         - - <400> SEQUENCE: 8                                                         - - caaaaccttg gccttctgg             - #                  - #                      - # 19                                                                   - -  - - <210> SEQ ID NO 9                                                   <211> LENGTH: 30                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: Primer         - - <400> SEQUENCE: 9                                                         - - tggccagcta gtctggtttg gttttctgga         - #                  - #               30                                                                      - -  - - <210> SEQ ID NO 10                                                  <211> LENGTH: 30                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: Primer         - - <400> SEQUENCE: 10                                                        - - ggagcatatt aaggtggaca gaagcagcag         - #                  - #               30                                                                      - -  - - <210> SEQ ID NO 11                                                  <211> LENGTH: 37                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: Primer         - - <400> SEQUENCE: 11                                                        - - attcagctac ttacagtgag ttggaagacc caggcgg      - #                       - #      37                                                                      - -  - - <210> SEQ ID NO 12                                                  <211> LENGTH: 36                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: Primer        - - <400> SEQUENCE: 12                                                        - - cacgtgattc ttataaatca atgggaggag acaatt      - #                  -     #       36                                                                    __________________________________________________________________________

What is claimed is:
 1. A method for diagnosing an individual as havingatopy or a predisposition thereto, which comprises demonstrating in theindividual the presence or absence of either: (i) a variant form of thegene which codes for the beta sub-unit of the high affinity receptor forIgE (FcεRIβ), the variant form comprising a variation in exon 7 whichencodes a glycine at amino acid residue 237 instead of glutamic acidwhich appears at residue 237 in individuals without atopy; or (ii) apolymorphic form of the amino acid sequence for FcεRIβ, the polymorphicform comprising glycine at amino acid residue 237 instead of glutamicacid which appears at residue 237 in individuals without atopy.
 2. Amethod according to claim 1 which comprises the steps of:obtaining asuitable tissue sample from the individual; preparing from the tissuesample a nucleic acid sample; and probing or analysing the nucleic acidsample for the presence or absence of said variant form of the genewhich encodes FcεRIβ.
 3. A method according to claim 2 wherein prior tosaid probing or analysis, said gene which encodes the variant form ofthe FcεRIβ gene is amplified.
 4. A method according to claim 3 whereinamplification is by PCR.
 5. A method according to claim 4 wherein thePCR amplification is carried out using a pair of oligonucleotide primersand a first member of the primer pair comprises a nucleotide sequencewhich hybridises under suitably stringent conditions to a substantiallycomplementary nucleotide sequence which is proximal to, and 5' of, thecodon for amino acid residue number 237 and the second member of theprimer pair comprises a nucleotide sequence which hybridises undersuitably stringent conditions to a substantially complementarynucleotide sequence which is proximal to, and 3' of, the codon for aminoacid residue number
 237. 6. A method according to claim 3 wherein theamplification is carried out using a pair of oligonucleotide primers anda member of the primer pair comprises a nucleotide sequence which itselfhybridises under suitably stringent conditions to the sequence ofnucleotides which give rise to said variant gene, such thatamplification will only proceed when said variant gene is present in thesample.
 7. A method according to claim 2 which comprises probing thenucleic acid sample with an oligonucleotide hybridisation probe whichunder suitable conditions of high stringency only shows substantialhybridisation to said variant gene and not to the equivalent gene inindividuals without atopy, such that hybridisation only occurs when thesaid variant gene is present in the sample.
 8. A method according toclaim 2 wherein the analysis of the nucleic acid sample for the presenceor absence of said variant gene is carried out by direct sequencing ofthe gene sequence which encodes the E237G polymorphism, and wherein thenucleotide sequences for 5' to 3' and 3' to 5' sequencing are amplifiedprior to said direct sequencing.
 9. A method according to claim 2wherein the analysis of the nucleic acid sample for the presence orabsence of said variant gene is carried out by single strandedconformational polymorphism (SSCP) analysis.
 10. A method according toclaim 1 wherein the presence or absence of the polymorphic form of theamino acid sequence for FcεRIβ is established by use of a substancewhich comprises an antibody binding domain which is specific for thepolymorphic form.
 11. A method according to claim 10 wherein thesubstrate comprises an antibody.
 12. A method according to claim 11wherein the antibody is either monoclonal or polyclonal.
 13. Anoligonucleotide hybridization probe for diagnosing an individual ashaving atopy or a predisposition thereto, which probe comprises asequence of nucleotides such that under suitably stringent conditionsthe probe: (i) specifically binds to a variant form of the gene whichcodes for FcεRIβ the variant form comprising a variation in exon 7 whichbinds to said probe and encodes a glycine at amino acid residue 237instead of glutamic acid which appears at residue 237 in individualswithout atopy; and (ii) fails to show significant hybridization togenetic material derived from individuals lacking said variant form ofsaid gene.
 14. A pair of oligonucleotide primers for amplification of avariant form of the gene which codes for FcεRIβ, for diagnosing anindividual as having atopy or a predisposition thereto, wherein thevariant form comprises a variation in exon 7 which encodes a glycine atamino acid 237 instead of glutamic acid which appears at residue 237 inindividuals without atopy; wherein a single member of the primer paircomprises a nucleotide sequence which itself hybridises under suitablestringent conditions to the sequence of nucleotides which give rise tosaid variant gene such that an amplification reaction employing such amember of a primer pair will only proceed where said variant gene ispresent in a sample.
 15. A pair of primers according to claim 14 whichis an amplification system function to amplify said variant form, butfail to significantly amplify genetic material derived from individualswithout atopy.
 16. An assay kit which comprises an oligonucleotidehybridisation probe according to claim
 13. 17. An assay kit whichcomprises a pair of oligonucleotide primers according to any one ofclaims 14 or
 15. 18. An assay kit which comprises an oligonucleotidehybridisation probe comprising a sequence of nucleotides such that undersuitably stringent conditions the probe: (i) specifically binds to avariant form of the gene which codes for FcεRIβ the variant formcomprising a variation in exon 7 which binds to said probe and encodes aglycine at amino acid residue 237 instead of glutamic acid which appearsat residue 237 in individuals without atopy; and (ii) fails to showsignificant hybridization to genetic material derived from individualslacking said variant form of said gene along with a pair ofoligonucleotide primers according to any one of claims 14 or
 15. 19. Amethod for diagnosing an individual as having atopy or a predispositionthereto, which comprises demonstrating in the individual the presence orabsence of either: (i) a variant form of the gene which codes for thebeta sub-unit of the high affinity receptor for IgE (FcεRIβ), thevariant form comprising a variation in exon 7 at a nucleotidecorresponding to nucleotide 7297 of the nucleic acid sequence depositedunder GenBank/Embl accession number M89796, which variation causes thecodon in which it appears to encode glycine instead of glutamic acidwhich is encoded by the equivalent codon in individuals without atopy;or (ii) a polymorphic form of the amino acid sequence for FcεRIβ, thepolymorphic form comprising glycine at the amino acid residue whichcorresponds to the codon of the nucleic acid sequence deposited underGenBank/Embl accession number M89796 which includes nucleotide 7297,instead of glutamic acid which appears at that residue in individualswithout atopy.
 20. A method according to claim 19 which comprises thesteps of:obtaining a suitable tissue sample from the individual;preparing from the tissue sample a nucleic acid sample; and probing oranalyzing the nucleic acid sample for the presence or absence of saidvariant form of the gene which encodes FcεRIβ.
 21. A method according toclaim 20 wherein prior to said probing or analysis, said gene whichencodes the variant form of the FcεRIβ gene is amplified.
 22. A methodaccording to claim 21 wherein amplification is by PCR.
 23. A methodaccording to claim 22 wherein the PCR amplification is carried out usinga pair of oligonucleotide primers and a first member of the primer paircomprises a nucleotide sequence which hybridizes under suitablystringent conditions to a substantially complementary nucleotidesequence which is proximal to, and 5' of, the codon for amino acidresidue number 237 and the second member of the primer pair comprises anucleotide sequence which hybridizes under suitably stringent conditionsto a substantially complementary nucleotide sequence which is proximalto, and 3' of, the codon for amino acid residue number
 237. 24. A methodaccording to claim 21 wherein the amplification is carried out using apair of oligonucleotide primers and a member of the primer paircomprises a nucleotide sequence which itself hybridizes under suitablestringent conditions to the sequence of nucleotides which give rise tosaid variant gene, such that amplification will only proceed when saidvariant gene is present in the sample.
 25. A method according to claim20 which comprises probing the nucleic acid sample with anoligonucleotide hybridization probe which under suitable conditions ofhigh stringency only shows substantial hybridization to said variantgene and not to the equivalent gene in individuals without atopy, suchthat hybridization only occurs when the said variant gene is present inthe sample.
 26. A method according to claim 20 wherein the analysis ofthe nucleic acid sample for the presence or absence of said variant geneis carried out by directing sequencing of the gene sequence whichencodes the E237G polymorphism, and wherein the nucleotide sequences forsequencing are amplified prior to said direct sequencing.
 27. A methodaccording to claim 20 wherein the analysis of the nucleic acid samplefor the presence or absence of said variant gene is carried out bysingle stranded conformational polymorphism (SSCP) analysis.
 28. Amethod according to claim 19 wherein the presence or absence of thepolymorphic form of the amino acid sequence for FcεRIβ is established byuse of a substance which comprises an antibody binding domain which isspecific for the polymorphic form.
 29. A method according to claim 28wherein the substance comprises an antibody.
 30. A method according toclaim 29 wherein the antibody is either monoclonal or polyclonal.